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Table of Contents
Vinegar Report................................................................................................................................2
Introduction.......................................................................................................................................2
Aim....................................................................................................................................................5
Objectives........................................................................................................................................5
Materials and Method.....................................................................................................................5
Results.............................................................................................................................................8
Titration...........................................................................................................................................10
Calculations...................................................................................................................................11
DISCUSSION................................................................................................................................13
CONCLUSION..............................................................................................................................14
References.....................................................................................................................................14

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 Vinegar Report

Introduction

Vinegar is derived from the French words vin (wine) and aigre (sour). It is a sour and sharp
liquid which can be used as a condiment or flavorant and food preservative. Vinegar is made
from various sugary and starchy materials by alcoholic and subsequent acetic fermentation
(WS, 1958). It is an aqueous solution made by a double fermentation process, first alcoholic
and then acetous. It is the product made from the conversion of ethyl alcohol which is
derived from yeast in yeast fermentation to acetic acid by a genus of bacteria, Acetobacter
(Gullo, 2008). Therefore, vinegar can be produced from any alcoholic material from alcohol-
water mixture to various fruits (J & G, 1967). Its colour and aroma are greatly dependent on
the material which it is made.
Vinegar has a pH range of 2-3; it includes around 5% acetic acid in water, as well as various
quantities of coloring matter, salts, and a few other fermentation products. Fermented ciders,
fruit juices, and other fermented alcoholic solutions obtained from barley malt, hydrolyzed
cereals, and starches can also be used to make vinegar. Different vinegars are made by
different items, and there are three different sorts of vinegars. Cider vinegar, for example, is
made from fermented apples. Traditional vinegars are produced through a slow fermentation
process that can take weeks or even months. Distilled alcohol is used to make white vinegar.
White grapes are used to make balsamic vinegar (Alsono et al, 2000)
It can be used in culinary such as pickling processes and other salad dressings. It is also an
ingredient in sauces such as mustard and ketchup. Vinegar also has medical uses such as
remedies and treatments. Rust can be removed from steel and iron using vinegar. Vinegar
can also be used as a cleaning product for example, white vinegar which is used as a
household cleaning agent (Kalaichelvan et al, 2007). It has advantageous effects on human
digestive tract and reducing effect on blood sugar level as well as cholesterol level. Obtained
from fermenting dilute alcoholic liquids, from alcohol-water mixtures to various fruit wines,
vinegar may also become a natural and safe source of destroying harmful germs.
The preparation of the raw material is a key stage in vinegar production. This step is
necessary to obtain the fermentable sugar and juice solution for acetification. The
processing varies according to the raw material utilized. Microbial species involved in
fermentations may range from yeast and lactic acid bacteria (LAB) to molds and acid
acetic bacteria (AAB) depending on the raw material used. Yeasts and AAB are the most
common microorganisms found in vinegar production. The former is in charge of alcoholic
fermentation by transforming fermentable sugars into ethanol, while the latter is required for
acetication, it oxidize the ethanol into acetic acid. Vinegar bacteria are members of the
Acetobacter, gluconacetobacter or gluconabacter genera. They are gram negative, catalase
positive, oxidase negative bacteria.
There are three methods of producing vinegar namely, Orleans method, generator method
and submerged method. The Orleans methods which is generally accompanied by the
generator method, is one of the oldest methods of vinegar production. In this method wine is
left in open vats to accumulate acetic acid bacteria from the atmosphere which aid in
converting the wine into vinegar, this method does not require inoculum it rely on the
microorganisms present in the atmosphere but in some industries an inoculum of acetic acid

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bacteria is inoculated to the wine by adding a small amount of vinegar (Tan, 2005). Wooden
barrels are stacked on their sides, with bungholes drilled on the top side and ends. A stopper
is placed on the topside. Long-necked funnels attached into holes poured the alcohol into
the barrel. Long-necked funnels placed into bungholes are used to pour alcohol into the
barrel. The barrel is filled to a level that is just below the end holes. For several months, the
barrels are left to rest. The temperature is kept at 29 degrees Celsius. By inserting a spigot
into the interior perforations and sucking liquid out, samples are taken on a regular basis.
The spigot is used to drain the alcohol once it has totally converted to vinegar. For the next
batch, around 15% of the liquid is left in the barrel (Kalaichelvan et al, 2007).
In generator method, a vinegar generator is set up as shown in figure 1. It consists of an
assemblage of 3 containers linked by tubes. The ethanol solution is poured on the first
container, the reservoir. The solution will be transferred to the other bottle via a drip tube
that has a flow regulator which regulate the pace at which the solution flows from the
reservoir to the second bottle. The second container is filled with any non-compacting
material (wood shavings charcoal or coke). It has wholes that allows a constant flow of
oxygen during the process and it is connected to the collector bottle via a drip tube. Once the
solution reaches the collector bottle it is poured back in the reservoir again, this process has
to be repeated for few weeks.

Reservoir

Drip tube

Wood shavings

Collector bottle

Figure 1: Vinegar generator

At the industry scale vinegar is produced using the submerged method. Submerged
fermentation is a process that involves the development of microorganisms in a liquid broth,
this liquid broth can be juice, sugar, or wine. During the submerged process bacteria are
constantly submerged in liquid and pure oxygen is pumped at the bottom of the stainless-
steel tanks. Strictly aerobic acetic acid bacteria are used for submerged fermentation,
converting ethanol into acetic acid (Gullo, et al, 2014).
It is important to access the composition of the acetic acid present in the vinegar production.
Acetic acid (CH3COOH) is one of the simplest organic carboxylic acid. This colourless weak
acid is characterized by distinctive sour taste and pungent smell. Nowadays, this acid is
considered as one of the key intermediate for many industries including: chemical,
detergent, wood and food industries. Currently, the production of acetic acid is carried out by
chemical means using petrochemical feedstock or by the traditional approach of
fermentative alcohol conversion using specific type of acetic acid bacteria.

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Vinegar is made from mostly acetic acid and some parts water. Vinegar bacteria, also called
Acetic Acid Bacteria (AAB).

Anaerobic Aerobic
Glucose + yeast = Ethanol+ AAB= Acetic acid
The concentration of acetic acid ranges between 4 to 8% by volume for table vinegar is
usually 5%. The acetic acid that makes up the vinegar is prepared from the fermentation of
ethanol alcohol by acetic acid bacteria of the family Acetobacter which are distinct by their
ability to ferment ethyl alcohol into acetic acid solution by oxidative reaction. Most bacteria
strains found in vinegar factories can oxidize acetic acid to CO2 and H2O (over-oxidation),
and so belong to the Acetobacter genus (Alson et al, 2004).
Vinegar's major ingredient is acetic acid. It is a carbon-based organic acid with a single
ionisable proton, making it a member of the larger Carboxylic acid class of organic acids,
which includes organic compounds having a –COOH functional group. Titration is an
analytical procedure used in laboratories to determine the concentration of a solution or a
reactant when the concentration of another solution is known. Acid-base titration is a
technique for determining the concentration of a basic solution given the concentration of an
acidic solution, and vice versa. By titrating the Acetic Acid with the strong base Sodium
Hydroxide, the percentage of Acetic Acid (CH3CO2H) in Vinegar can be measured (NaOH).
The phenolphthalein indicator, an acid-base indicator that changes color from clear to pink
as it transitions from its acidic to basic state, will be used to determine the titration's end
point (Chen, 2009).
Ethanol is a clear, colorless liquid with a distinctive, pleasant odor. It has a pleasant taste in
a dilute aqueous solution, but a scorching taste in a more concentrated solution. Ethanol
(CH3CH2OH) is an alcohol, a class of chemical substances with a hydroxyl group (-OH)
attached to a carbon atom in its molecules. Ethanol has a density of 0.789g/ml at 20°C,
melts at -114°C, boils at 78.5°C, and melts at -114°C. Its low freezing point has made it
useful as fluid thermometers for temperatures below -40°C, mercury freezing point, and
other low temperature applications, such as antifreeze in vehicle radiators (Sengun et al,
2000).
The Acetobacter agar is used as a maintenance media for glucose positive Acetobacter
species (in text). It is a selective and differential medium which permit isolation of acetic acid
bacteria and differentiation between Gluconobacter and Acetobacter. Isolation is achieved
by inhibition of the growth of any other bacteria : acidification and to inhibit yeast and mold
growth And/or addition of grams of brilliant green or sodium desoxycholate to minimize
interference by other acid tolerant bacteria. Whereas differentiation is based on the
preferential oxidation of carbon sources.

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Aim

To produce vinegar using a simple vinegar generator.

Objectives

1. To construct and operate a simple vinegar generator

2. To determine acetic acid concentration and percentage by titration
3. To perform spread plate technique

Materials and Method

Part One: Construction of Vinegar Generator.

Materials
 Vinegar
 Bostik clear glue
 A drip tube
 Flow regulator
 Retort stand and clamp
 Wood shavings
 2 x 2 Litre plastic cold drink bottles
 5 Litre plastic bottle
 10% of 1,5 litre ethanol solution

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Procedure
Out of the 3 bottles used, 1 bottle served as the ethanol reservoir and the bottom of the
bottle was cut open. The bottle was attached to the clamp. The second bottle was filled was
filled with the wood shavings and soaked in vinegar. The excess vinegar was poured off. In
the top half of bottle holes were made. The two bottles were connected with the drip tubes
and the flow regulator were placed between the bottles. A second drip tube led from the
second bottle with wood shavings to the 5 Litre plastic bottle. All the joints were sealed with
the epoxy glue. 10% of ethanol solution was poured into the reservoir bottle and the flow
was adjusted so as to allow the solution to drip slowly onto the wood shavings. Once the
solution collected in the 5 Litre bottle, it was poured back to the reservoir – and this whole
process was of recycling the vinegar for a period of 4 weeks which allowed the concentration
of acetic acid to increase. Therefore, the pH of the product was measured at weekly intervals
since from the beginning. This was done so as to determine the acid produced.
In the results section the graph of the differences in pH was plotted. Notes of all changes in
appearance, smell, taste of the product was made.

Part Two: Determination of Acetic Acid (Vinegar) Concentration and % by Titration.

Requirements:
 2 x 250 ml beakers
 50 ml Burette
 25 ml Pipette
 250 ml volumetric flask
 Wash bottle
 0,1 mol.dm-3 NaOH standardised solution
 Phenolphthalein Indicator
 3 x 250 ml Erlenmeyer flasks

Procedure
25cm2 vinegar was pipetted into a 250cm2 volumetric flask and was further diluted to the
mark with distilled water. The 25cm2 of the same solution was pipetted in an Erlenmeyer
flask. The 2 drops of the phenolphthalein indicator were added and was titrated against the
standardised 0.1M NaOH solution. This procedure was in triplicate.

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Part 3: Isolation of Organisms from the Vinegar Generator

Requirements:
 Acetobacter agar plate.
 Sample of vinegar from the Generator.

Procedure:
A loopful of the vinegar was inoculated from the group’s generator onto an Acetobacter agar
plate. The plate was incubated at room temperature for 48 hours.

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Results

Table 1 Showing pH values that were recorded over 5 weeks in Lab F104

Figure 2 Showing the recycling of the vinegar generator in Lab F104

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Figure 3 Showing 3 Erlenmeyer flasks used for titration. 25ml of diluted vinegar was titrated
against the standardized 0.1 M NaOH solution. A pink colour was observed in all 3
Erkenmeyer flasks.

Figure 4 Showing Acetobacter agar plate after being incubated at room temperature for 48
hours . No growth was observed . A small bubble was observed on the plate.

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Titration

Table 2 Showing titration results

Titration 1 2 3

Volume of diluted 25mL 25 mL 25 mL

vinegar

Final burette 2.6 5.5 8.2

reading

Original burette 0.0 2.6 5.5

reading

Volume NaOH 2.6 2.9 2.7

Average Volume 2.73ml

NaOH

Concentration 0.011mol/L
diluted vinegar

Concentration 0.11 mol/L

original vinegar

% CH3COOH 6.6%

The calculated concentration of diluted vinegar was 0.011 mol/L and of original vinegar was
0.11 mol/L. the calculated percentage of acetic acid (CH3COOH) was 6.6%

Table 3 Showing pH values of Vinegar results

Duration pH Observations

Week 1 3.8 Clear, no smell

Week 2 3.3 Yellow, acidic smell

Week 3 3.4 Yellow, acidic smell

Week 4 3.5 Yellow, acidic smell

From the first week the pH dropped from 3.8 to 3.3 in the second week and the colour
changed from clear to yellow and the smell changed from no smell to acidic smell. From the
second week to the fourth week the pH increased with 0.1 each week, the colour remained
yellow and the smell stayed acidic.

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Figure 5 Showing Graphical representation of the pH scale of the product after four weeks.

The pH scale versus weekly intervals

3.9

3.8

3.7

3.6

3.5
pH scale

3.4

3.3

3.2

3.1

3
week 1 week 2 week 3 week 4
Weekly Intervals

Calculations

1. Concentration of diluted acetic acid

CV × NaOH
C(CH3COOH) =
V (CH 3 COOH )
2.73 mL ×0.1 mol /L
=
25 mL
=0.011 mol/L

2. Concentration of the original acetic acid

C1V1=C2V2
0.011mol / L× 250 mL
C1=
25 mL
C1=0.11mol/L

3. % CH3COOH

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 ( M ×V ×C ) CH 3 COOH
m(CH3COOH) =
1000
60.05196 g /mol ×25 ml × 0.11mol / L
=
1000
m = 0.165g

m ( CH 3 COOH )
Therefore % CH3COOH= ×100
m ( sample )
0.165
= ×100
25
=6.65%

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DISCUSSION

After inoculating a loopful of vinegar onto Acetobacter Agar plate, and incubated for 48
hours, no growth was observed. A possible explanation for this could be that the
Acetobacter species that was carrying out the conversion of ethanol to acetic acid did not
utilize mannitol. Other experiments of this kind use the Standard Medium Glucose, Yeast
extract and Calcium Carbonate (commonly called GYC Agar) for the isolation of Acetobacter
in vinegar. GYC Agar, described by Swings (1992), detects the presence of acid-producing
microorganisms and is regarded as “standard growth medium” for acetic acid bacteria.
Dextrose is the fermentable carbohydrate providing carbon and energy. Yeast extract is a
source of vitamins. Growth on this medium, Acetobacter will produce clear zones or halos
around colonies because the acid being produced will neutralize the CaCO 3 in the medium
(Madigan, Martinko, & Brock, 2006)
The conversion of ethanol to acetic acid consists of two steps, during the first step, ethanol is
converted into acetaldehyde (ethanal), after which the acetaldehyde is converted to acetic
acid (Burggraaf, 2017). In the vinegar generator, one 2-liter bottle was filled with wood
shavings which were a natural source of acetic acid bacteria. These bacteria belong to the
family Acetobacteraceae, which includes several genera and species. The main species
responsible for the production of vinegar belong to the genera Acetobacter,
Gluconacetobacter, Gluconobacter and Komagataeibacter because of their high capacity to
oxide ethanol to acetic acid and high resistance to acetic released into the fermentative
medium (Gomez, De Fatima, De Freitas, Castro-Gomez, & Spinosa, 2018).
At the beginning of the 4-week cycle, during which the produced acetic acid was
continuously recycled, the starting pH was recorded to be 3.8. The final pH reading was
recorded to be 3.5. Essentially, there was a significant drop of 0.3 pH units since the start of
the experiment, this was because there was an increase in the concentration of acetic acid
produced during that period.
Alcohol was converted, with the pre+
sence of oxygen, to acetic acid by acetic acid bacteria. For this experiment, an Acetobacter
Agar was used to isolate the microorganism that was responsible for the conversion of
ethanol to acetic acid. Acetobacter Agar (Mannitol) was used as a maintenance medium for
Acetobacter species utilizing mannitol (Gherna, Pienta, & Cote, 1992).

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CONCLUSION

The percentage of acetic acid that was calculated to be 6.63% which was in the normal
range of 5-8% which shows that the vinegar was good for consumption. There was no
growth on the Acetobacter Agar plate after incubation period of 48hours which could be due
to human error. It might be found that the incubation duration was not enough for growth to
be observed

References

Burggraaf, W. (2017). Vinegar Production. Safe Food Factory.

Gherna, R., Pienta, P., & Cote, R. (1992). Catalogue of Bacteria and Bacteriophages, 18th
Ed. Rockville: American Type Culture Collection.
Gomez, R. J., De Fatima, M., De Freitas, M., Castro-Gomez, R. J., & Spinosa, W. A. (2018).
Acetic acid bacteria in the Food Industry: Systematics, Characteristics and Applications.
Food Technology and Biotechnology, 138-151.
Madigan, M. T., Martinko, J. M., & Brock, T. D. (2006). Brock Biology of Microorganisms,
11th Ed. Upper Saddle River: Pearson Prentice Hall.

Bourgeois, J.F. and Barja, F., 2009. The history of vinegar. Archives des sciences, pp.147-
160.
Tan, S.C., 2005. Vinegar fermentation.
Gullo, M., Verzelloni, E. and Canonico, M., 2014. Aerobic submerged fermentation by acetic
acid bacteria for vinegar production: process and biotechnological aspects. Process
Biochemistry, pp.1571-1579.

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